The formation of new blood vessels either from differentiating endothelial cells during embryonic development (vasculogenesis) or from pre-existing vessels during adult life (angiogenesis) is an essential feature of organ development, reproduction, and wound healing in higher organisms.
Therapeutic angiogenesis is an effective means to treat patients suffering from a disease or a disorder leading to tissue ischemia.
Treatment of ischemia, using non-surgical therapy, has become possible with the discovery of angiogenic factors favouring formation of new blood vessels. Several candidate angiogenic factors have been described so far which were the subject of clinical trials.
Enthusiasm has however been hampered by series of negative clinical outcomes. Regarding VEGF for example, despite the potent angiogenic effects of this factor, its expression did not efficiently improve muscle blood flow in patients. This was explained by the formation of leaky vascular lacunae and arteriovenous shunts interfering with the downstream microcirculation.
Endothelial progenitor cells (EPCs) have been identified in adult human peripheral blood, in bone marrow and in cord blood (Asahara T, Murohara T, Sullivan A, Silver M, van der Zee R, Li T,Witzenbichler B, Schatteman G, Isner J M. Isolation of putative progenitor endothelial cells for angiogenesis. Science. 1997 275:964-7). Circulating EPCs participate in postnatal neovascularization after mobilization from the bone marrow. Transplantation of culture-expanded EPCs, obtained either from blood or from autologous bone marrow mononuclear cells, was found to be able to augment ischemia-induced neovascularization in vivo.
The use of cultured cells as a therapeutic approach in patients is however considerably limited by the small proportion of EPCs in the peripheral blood, the necessity of harvesting a large amount of bone marrow to isolate a sufficient number of EPCs, and the heterogeneity of the recovered EPCs.
Despite their drawbacks, the use of angiogenic growth factors thus remains to date the primary strategy of therapeutic angiogenesis for the treatment of patients, such as patients presenting with severe peripheral arterial disease (also called peripheral vascular disease) or ischemic heart disease.
CD146, also known as MCAM, MUC18, or Mel-CAM, is a component of the endothelial junction which belongs to the immunoglobulin superfamily (Bardin N, Anfosso F, Massé J M, Cramer E, Sabatier F, Le Bivic A, Sampol J, Dignat-George F. Identification of CD146 as a component of the endothelial junction involved in the control of cell-cell cohesion. Blood. 2001; 98:3677-84). As a member of such a family, it consists in five Ig domains, a transmembrane domain, and a cytoplasmic region.
CD146 is mainly known to occur in two distinct forms differing by the length of their cytoplasmic domain: a long isoform (herein identified as “long CD146”) and a short isoform (herein identified as “short CD146”), both present in the membrane of cells, mainly endothelial cells.
CD146 is involved in the control of cell and tissue architecture, as demonstrated by the regulation of its expression during endothelium monolayer formation, its involvement in the control of paracellular permeability (Bardin N, Anfosso F, Massé J M, Cramer E, Sabatier F, Le Bivic A, Sampol J,Dignat-George F. Identification of CD146 as a component of the endothelial junction involved in the control of cell-cell cohesion. Blood. 2001; 98:3677-84) and its colocalization with the actin cytoskeleton (Anfosso F, Bardin N, Vivier E, Sabatier F, Sampol J, Dignat-George F. Outside-in signaling pathway linked to CD146 engagement in human endothelial cells. J Biol Chem. 2001; 276:1564-9).
Membranous CD146 has been reported to promote tumor growth, angiogenesis, and metastasis in human melanoma. Membranous CD146 expression levels and distribution are closely associated with tumor progression and onset of metastasis in human malignant melanoma. Anti-membranous CD146 antibodies have been described as capable of significantly inhibiting the growth and metastasic properties of human melanoma cells in nude mice (Mills L, Tellez C, Huang S, Baker C, McCarty M, Green L, Gudas J M, Feng X, Bar-Eli M. Fully human antibodies to MCAM/MUC18 inhibit tumor growth and metastasis of human melanoma. Cancer Res. 2002; 62:5106-14.). Membranous CD146 has been shown to display angiogenic properties, both in an in vitro model of human umbilical vein endothelial cells (HUVEC) (Kang Y, Wang F, Feng J, Yang D, Yang X, Yan X. Knockdown of CD146 reduces the migration and proliferation of human endothelial cells. Cell Res. 2006; 16(3):313-8) and in in vivo models of chicken chorioallantoic membrane (CAM) assays and tumor growth in mice (Yan X, Lin Y, Yang D, Shen Y, Yuan M, Zhang Z, Li P, Xia H, Li L, Luo D, Liu Q, Mann K, Bader B L. A novel anti-CD146 monoclonal antibody, AA98, inhibits angiogenesis and tumor growth Blood. 2003;102:184-91). mAb AA98 has been shown by Yan et al. to display a remarkably restricted immunoreactivity against intratumoral vasculature compared with blood vessels of normal tissues.
Finally, inventors recently showed that CD146 was involved in the regulation of monocytes transendothelial migration (CD146 and its soluble form regulate monocytes transendothelial migration. Arteriosclerosis, thrombosis and Vascular Biology, 2009; 29: 746-53).
Different localisations and functional differences have been identified in the literature for the two membranous isoforms of chicken CD146. In one study, authors analyzed chicken CD146 targeting in polarized epithelial Madin-Darby canine kidney (MDCK) cells using CD146-GFP chimeras, to identify the respective role of each isoform. They showed by confocal microscopy that short CD146 and long CD146 were addressed to the apical and basolateral membranes, respectively (Guezguez B, Vigneron P, Alais S, Jaffredo T, Gavard J, Mège R M, Dunon D. A dileucine motif targets MCAM-1 cell adhesion molecule to the basolateral membrane in MDCK cells. FEBS Lett. 2006; 580:3649-56). In another study, the same group showed that long CD146 promoted rolling via microvilli induction in lymphocytes and displayed adhesion receptor activity, suggesting its involvement in the recruitment of activated T cells to inflammation sites (Guezguez B, Vigneron P, Lamerant N, Kieda C, Jaffredo T, Dunon D. Dual role of melanoma cell adhesion molecule (MCAM)/CD146 in lymphocyte endothelium interaction: MCAM/CD146 promotes rolling via microvilli induction in lymphocyte and is an endothelial adhesion receptor. J Immunol. 2007; 179:6673-85).
The existence of a soluble form of CD146 has been discovered initially from a western blot and its possible role as a competitive inhibitor of the CD146 membrane-bound form has been suggested (Bardin N, Francès V, Combes V, Sampol J, Dignat-George F. CD146: biosynthesis and production of a soluble form in human cultured endothelial cells. FEBS Lett. 1998; 421:12-4).
However, until now, the soluble form has not been structurally or functionally characterized. A breakthrough results from the inventors' discovery that biologically active forms of human CD146 exist not only as membrane-bound forms but also as a soluble form present in the human serum. Inventors first suggested that changes in sCD146 levels may be related to physiopathological conditions associated with alteration in endothelial barrier integrity such as permeability, leukocyte transmigration or angiogenesis (N. Bardin, F. Anfosso, V. Combes, J. Nedelec, I. Besson-Faure, P. Brunet, V. Moal, J. Sampol, and F. Dignat-George. Soluble CD146, a junctional endothelial adhesion molecule, is increased in vascular disorders, Workshop K endothelial cells; DK, vol. 55, no. SUPPL. 01, 1 Jan. 2000, page 63, ISSN: 0340-6245) and then described increased levels of sCD146 in the plasma of patients with chronic renal failure (Bardin N, Moal V, Anfosso F, Daniel L, Brunet P, Sampol J, Dignat-George F. Soluble CD146, a novel endothelial marker, is increased in physiopathological settings linked to endothelial junctional alteration, Thromb Haemost. 2003; 90:915-20). Inventors now herein describe, for the first time, the structure of said soluble forms and demonstrate (see in particular in vivo experimental results herein provided) the therapeutic properties of the human soluble CD146, in particular the angiogenic properties thereof, in contradiction with the art suggestions and in particular with previous observations from Wu Guang-JER et al. (see Wu Guang-JER et al.: “Soluble METCAM/MUC 18 blocks angiogenesis during the in vivo tumor formation of human prostate cancer LNCaP cells.” Proceedings of the American Association for cancer research annual meetings, vol. 47, April 2006, page 59 n & 97TH annual meeting of the AACR; Washington DC, USA, Apr. 01-05, 2006, ISSN: 0197-016X).
In the literature, different soluble receptors, as soluble EphB4 or soluble Notch1, have been shown to act as endogenous inhibitors of angiogenesis, acting as traps for their ligand. This is also the case for the soluble form of VEGFR2 which blocks the angiogenic effect of VEGF (Holash J, Davis S, Papadopoulos N, et al. VEGF-Trap: a VEGF blocker with potent antitumor effects. Proc Natl Acad Sci U S A. 2002; 99: 11393-8.). In contrast, other soluble molecules have been shown to act as activators of angiogenesis, such as the soluble N-cadherin fragment (Derycke L, Morbidelli L, Ziche M, et al. Soluble N-cadherin fragment promotes angiogenesis. Clin Exp Metastasis. 2006; 23: 187-201) or the soluble CD40 ligand (Melter M, Reinders M E, Sho M, et al. Ligation of CD40 induces the expression of vascular endothelial growth factor by endothelial cells and monocytes and promotes angiogenesis in vivo. Blood. 2000; 96: 3801-8.). The reason for the observed opposite effects of soluble molecules, inhibitor or activator, is unknown but may result from distinct signalling pathways. Thus, one can hypothesize that soluble forms of receptor molecules may trap the ligand and inhibit the effect. In contrast, other soluble molecules, such as soluble CD146, result from a membrane protein shedding, and could serve as a ligand that activates its receptor.
Inventors in particular herein provide new tools, using the soluble form of CD146, improving the treatment of tissue ischemia while reducing deleterious side effects observed with classically used therapies. They herein demonstrate that the soluble form of CD146 fulfills key functions in the neovascularisation process.
Inventors herein characterize the human soluble form of CD146 (herein identified as “soluble CD146”) and identify amino acid sequences thereof usable in the context of a treatment. Inventors in particular describe its advantageous chemotactic and angiogenic effects on endothelial cells, in particular on endothelial progenitor cells (EPC). The human soluble form of CD146 is able to promote a therapeutic vasculogenesis and/or angiogenesis in a mammal subject, in particular in a human subject.
Other advantages of the products and compositions herein described are further indicated below.